Multiplexed bead-based analytical assays

ABSTRACT

Methods for performing bead-based analytical assays for detecting changes in abundance of target analytes in biological samples are disclosed. In an embodiment, a method involves eluting bead-captured analytes from a bead array under conditions that preserve spatial localization of the eluted analytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/932,732, inventor Vladislav B. Bergo, filed Jul. 18, 2020,which, in turn, claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application No. 62/876,060, inventor Vladislav B.Bergo, filed Jul. 19, 2019, the disclosures of both of which areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing XML which has beensubmitted electronically in XML file format and is hereby incorporatedby reference in its entirety. Said XML copy, created on Mar. 21, 2023,is named 84233B.xml and is 10,017 bytes in size.

FIELD

The embodiments disclosed herein relate generally to bead-based assaysand more specifically to measuring analytes in biological samples usingbead-based assays. The embodiments disclosed herein also relate toproteomics, protein quantification, post-translational modifications ofproteins, affinity separations, microarrays and mass spectrometry.

BACKGROUND

Detection, identification and quantification of multiple analytes inbiological samples is an important area of basic and applied biology. Inmany applications, the analytes are proteins and/or protein fragments,such as proteolytic peptides produced by enzymatic digestion ofprecursor proteins. Mass spectrometry (MS) is the key analyticalplatform for quantitative analysis of proteins and peptides.MS-dependent protein quantification methods can be classified as eitherlabel-free or label-based.

Recently there has been considerable progress in the MS-basedquantification methods that utilize detection from individual beads ormicrospheres. One example of such approach, termed “immuno-MALDI” or“iMALDI” is described in the U.S. Pat. No. 7,846,748 and severalpublications. The iMALDI approach is generally limited to detecting asingle analyte. On the other hand, recent U.S. Pat. No. 9,618,520 andU.S. patent application Ser. No. 13/369,939, Publication No. US2012-0202709 A1 describe various quantitative bead-based methods, whichare multiplexed, that is capable of measuring multiple distinctanalytes.

While analytical assays utilizing single beads can be readily adaptedfor use with label-based quantification methods, such as tandem masstags (TMT) and Stable Isotope Labeling by Amino acids in Cell culture(SILAC), developing label-free assays is more difficult. The challengesinclude optimizing the analyte binding capacity of individual beads andof the entire bead array, creating conditions that enable depletion ofone or more analytes from a sample and detecting the bead-capturedanalytes using MS.

Accordingly, there is still a need for methods and compositions thatwill enable analysis of proteins and peptides by MS in a bead arrayformat.

SUMMARY

In one aspect, the present specification describes methods of preparinga sample for analysis by MS. Some of the described methods includedepleting one or multiple analytes from the sample by incubating thesample with a bead array.

In another aspect, the present specification describes a bead array, inwhich individual reactive sites contain at least two distinct captureagents that are present in a specific ratio. A single reactive site ofsuch bead array is capable of binding at least two distinct analytesfrom a sample.

In yet another aspect, the present specification describes a method ofmeasuring abundance of an analyte in a sample. The method includes thestep of binding at least two distinct analytes from the sample to asingle reactive site of a bead array, such that one of the analytesbecomes substantially depleted from the sample while a certain amount ofthe other analyte remains in the sample. The bead-captured analytes aresubsequently measured by MS from individual reactive sites of the beadarray.

In yet another aspect, the present specification describes methods ofpreparing a bead array for analysis by MS. Some of the described methodsinvolve the step of continuously adding a solution that contains MSmatrix to a liquid-filled microwell, such that a rate, at which thematrix solution is being added to the microwell, is substantiallyequivalent to a rate, at which the solvent of the matrix solution isescaping from the microwell via evaporation. The described methodsenable adding the matrix solution to a liquid-filled microwell withoutsplattering or spilling.

In yet another aspect, the present specification describes a microarray,in which spots that contain or are suspected of containing biomoleculesare visibly distinct from spots that are devoid of biomolecules. Thelatter spots may form rows and/or columns, which are located on aperiphery of the microarray or separate distinct regions within themicroarray. The visibly distinct appearance of the spots that are devoidof biomolecules is used to identify such spots and exclude them from themicroarray analysis.

In yet another aspect, the present specification describes a microwellarray plate that includes a flat surface solid support, such as amicroscope slide and an elastomer gasket that contains an array ofthrough-holes. Distinct surfaces of the elastomer gasket have distinctadhesive properties. The gasket optionally contains a chamfer or anothervisual marking that allows identification of a more adhesive surface.

The methods and compositions described in this specification may beutilized to analyze various biological samples, including cell-freeprotein transcription-translation reactions, bacterial cells, mammaliancells, cell culture supernatants, animal models, xenografts, tissuebiopsies, biofluids such as serum, plasma and cerebrospinal fluid, andothers. The described methods and compositions may be utilized in abroad range of applications including basic research, pharmaceuticaldrug discovery and drug development, disease diagnostics andprognostics, biomarker discovery and validation, personalized medicine,precision medicine, systems biology and others.

DESCRIPTION OF FIGURES

The presently disclosed embodiments will be further explained withreference to the attached drawings, wherein like structures are referredto by like numerals throughout the several views. The drawings shown arenot necessarily to scale, with emphasis instead generally being placedupon illustrating the principles of the presently disclosed embodiments.

FIGS. 1A through 1D schematically depict various affinity bindingreactions that include incubating a sample with a bead array.

FIG. 1A schematically depicts an affinity binding reaction, in which asample contains a first (target) peptide and a second (reference)peptide and a binding capacity of a bead array is greater that an amountof the first peptide and lower than an amount of the second peptide inthe sample.

FIG. 1B schematically depicts an affinity binding reaction, in which asample contains two distinct peptides and a bead array contains distinctreactive sites that recognize these peptides.

FIG. 1C schematically depicts an affinity binding reaction, in whichsamples that contain different amounts of a particular peptide areincubated with two bead arrays.

FIG. 1D schematically depicts an affinity binding reaction, in which asample is consecutively incubated with two bead arrays.

FIG. 2A schematically depicts a microarray of spots that contain MSmatrix.

FIG. 2B schematically depicts a section of a microarray, in which spotsthat contain an analyte or are suspected of containing an analyte arevisibly distinct.

FIG. 3 is a photograph of a reusable microarray substrate.

FIG. 4 is a photograph of a microarray of spots that contain MS matrix.

DETAILED DESCRIPTION

The term “bead array” refers to a group that includes at least tworeactive sites. A bead array may be located in a container, such as amicrocentrifuge tube, or in a well of a multiwell plate, in which caseit may be referred to as a suspension bead array. Alternatively, a beadarray may be positioned on a solid support, in which case it may bereferred to as a planar bead array. A variation of a planar bead arrayis a bead array, in which individual reactive sites are positionedwithin size-matching microwells of a microwell array slide, with asingle microwell usually being dimensioned to hold no more than onereactive site.

The term “reactive site” refers to a combination of a bead and at leastone capture agent that is associated with the bead.

The term “capture agent” refers to a molecule or a molecular complexthat is capable of binding a compound. A singular form of the term“capture agent” may refer to a plurality of identical molecules or aplurality of identical molecular complexes. For example, it may refer toa plurality of identical antibody molecules.

The terms “target analyte” and “target” are used interchangeablythroughout the instant specification and generally refer to a bindingpartner of a capture agent. Singular forms of the terms “target analyte”and “target” may refer to a plurality of molecules, e.g. a plurality ofpeptide molecules.

The terms “peptide” and “polypeptide” are used interchangeablythroughout the specification and refer to a combination of at least twoamino acids that are linked by an amide bond, which is also known as apeptide bond.

The term “protein” refers to a molecule or a molecular complex thatcontains at least one polypeptide.

The terms “well” and “microwell” are used interchangeably throughout theinstant specification and refer to a topological feature such as a pitor a depression that is able to hold a liquid medium, a particle orboth.

The term “microarray” refers to a plurality of spatially separated spotsthat are positioned on a substantially flat surface of a solid support.Individual spots within a microarray may contain a matrix for massspectrometry.

In an embodiment, the instant specification describes a method forsubstantially depleting one or multiple peptide analytes from a sampleusing affinity capture of the analytes on individual reactive sites of abead array, optionally followed by MS detection of the captured analytesfrom individual reactive sites. The described method is useful forquantifying one or more analytes in two or more samples.

In reference to FIG. 1A, a sample 108 contains a first (target) peptide107 and a second (reference) peptide 106 that is structurally distinctfrom the first peptide. The sample is brought in contact with a beadarray, which contains a reactive site 101. The reactive site contains abead 102, a capture agent 104 that specifically recognizes the firstpeptide, and a distinct capture agent 103 that specifically recognizesthe second peptide. Contacting the sample with the bead array causes thefirst peptide and the second peptide to bind to the reactive site andtherefore to the bead array. The binding capacity of the bead array andthe duration of the contacting step are selected to cause the firstpeptide to become substantially depleted from the sample, e.g. less than50%, less than 40%, less than 30%, less than 20%, less than 10%, or lessthan 5% of the first peptide 107 remains in the sample 110 after thecontacting step whereas at least some amount e.g. more than 50% of thesecond peptide 106 remains in the sample 110. In other words, thebinding capacity of the bead array is selected to be: (i) approximatelyequal to or greater than an amount of the first peptide that is presentin the sample and (ii) approximately equal to or lower than an amount ofthe second peptide that is present in the sample. After the contactingstep, the reacted reactive site 109 contains the bound first and secondpeptides, which are then released individually from the reactive siteand measured using MS. The MS data is used to obtain a ratio of thefirst peptide to the second peptide in the reactive site. The ratio isthen used to determine the amount of the first peptide in the sample. Inan embodiment, the amount of the first peptide in the sample isdetermined quantitatively, e.g. 1.2±0.3 pmol. In an embodiment, theamount of the first peptide in the sample is determined as being eitherabove or below a specific pre-determined value, such as being greaterthan 1.2±0.3 pmol, or being lower than 100±25 fmol.

The method described above enables quantitative measurement of an amountof the target peptide that is present in the sample because asubstantial amount, e.g. more than 50%, more than 60%, more than 70%,more than 80%, more than 90%, or more than 95% of the target peptidefrom the sample is captured by the bead array and a signal from thetarget peptide is detected by MS along with a signal from the referencepeptide, the latter providing a reference, so that the intensity ofsignal from the target peptide can be readily compared to the intensityof signal from the reference peptide. The quantitative measure of anamount of the target peptide that is originally present in the sample isthe intensity of MS signal from the target peptide that is normalizedagainst the intensity of MS signal from the reference peptide. When twoor more samples are measured using the above-described method, adifference in the ratio of the target peptide signal to the referencepeptide signal observed between the samples is indicative of adifference in the amounts of the target peptide that is present in thecorresponding samples.

The sample 108 may be prepared using one of the known methods ofbottom-up proteomics, in which a biological material, such as culturedmammalian cells, bacterial cells, tissue biopsy or other is subjected tocell lysis, protein denaturation, disulfide bond reduction, cysteinealkylation and protein digestion, optionally followed by desalting andlyophilization of the produced proteolytic peptides. Sample preparationprotocols for bottom-up proteomics are well-known and may be found innumerous publications. A detailed protocol is also provided in thisspecification. Alternatively, the sample may be prepared using knownmethods of middle-down proteomics or top-down proteomics.

The target (first) peptide may be produced by enzymatic digestion of aprecursor protein that is originally present in the biological material.The target peptide may be selected to monitor abundance of the precursorprotein; alternatively, it may be selected to monitor a site-specificpost-translational modification (PTM) of the protein, such asphosphorylation, acetylation, methylation, glycosylation,ubiquitination, sumoylation, or other. The target peptide may be alsoselected to simultaneously monitor several PTMs that are locatedsufficiently close to each other in the sequence of the precursorprotein.

Alternatively, the target peptide may be obtained without performingcell lysis or protein digestion. For example, unfractionated serum andplasma are known to contain large numbers of circulating peptides. Otherexamples include secreted peptides that are released from culturedmammalian or bacterial cells into a cell culture medium.

The reference (second) peptide may be produced either by enzymaticdigestion of a protein that is originally present in the biologicalsample, by chemical synthesis, or by enzymatically digesting achemically synthesized precursor peptide. The reference peptide is notrequired to contain stable isotope-labeled amino acids and is notrequired to have an amino acid sequence that is substantially similar tothe sequence of the target peptide. Preferably the molecular weight ofthe reference peptide differs from the molecular weight of the targetpeptide by less than 5 kDa. The reference peptide may be introduced intothe sample before, during or after the protein digestion step. Thepurity of the synthetic reference peptide may vary, however in somecases the peptide purity of 90% or higher is preferred because higherpurity peptides generate “cleaner” mass spectra that contain fewer peaksthereby facilitating data interpretation.

The reactive site of the bead array contains a bead and at least twodistinct capture agents, e.g. antibodies that are bound to the bead. Inan embodiment, the bead is made of porous material such as agarose,cellulose or controlled pore glass and has magnetic properties, whichfacilitate handling of the bead array. The antibodies are preferablycovalently bound to the bead, either directly or via an adaptermolecule, such as Protein A, Protein G, Protein A+G, biotin-avidin, etc.The target peptide-specific antibody specifically recognizes an epitopethat exists in the target peptide and is thus able to specifically bindthe target peptide to the reactive site. The reference peptide-specificantibody specifically recognizes an epitope that exists in the referencepeptide and is thus able to specifically bind the reference peptide tothe reactive site. A molar ratio of the former antibody to the latterantibody in the reactive site is preferably greater than 1:1, forexample it may be greater than 3:1, greater than 5:1, greater than 10:1or greater than 20:1. The reactive site preferably contains between 100femtomoles and 5 picomoles of the target peptide-specific antibody andbetween 5 and 500 femtomoles of the reference peptide-specific antibody.

During the contacting step, the target peptide-specific antibody mayspecifically bind multiple additional peptides from the sample, if suchpeptides contain the recognized epitope. This effect enables on-beadmultiplexing and analysis of several target analytes from a singlereactive site. The additional peptides may arise from incompleteproteolytic digestion of the precursor protein, presence of PTMs,presence of protein isoforms or appearance of the epitope in otherwiseunrelated proteins. Preferably, the number of such peptides in thesample is limited, e.g. less than 20 distinct sequences, less than 15distinct sequences or less than 10 distinct sequences. Binding fewerdistinct peptide analytes to a reactive site may be accomplished by (i)selecting an antibody, which recognizes an epitope that is present in alimited number of proteins within the proteome, (ii) performing morecomplete digestion, i.e. minimizing the amount of partially digestedprotein fragments, (iii) selecting protein fragments that contain fewerPTMs, etc.

The binding capacity of a reactive site of a bead array for a particularanalyte is defined is the maximum amount of the analyte that mayspecifically bind to the reactive site. A reactive site of the beadarray described above contains two distinct antibodies and is thereforecapable of binding at least two distinct analytes: the target peptideand the reference peptide. Such reactive site may be characterized ashaving a binding capacity for the target peptide and a distinct bindingcapacity for the reference peptide. The analyte binding capacity of asingle reactive site may be assumed to be approximately equivalent to anamount of the analyte-specific antibody that is present in the reactivesite. For example, a reactive site, which contains 1 picomole of thetarget peptide-specific antibody and 50 femtomoles of the referencepeptide-specific antibody, is capable of specifically binding up toapproximately 1 picomole of the target peptide and up to approximately50 femtomoles of the reference peptide. The analyte binding capacity ofa reactive site may be experimentally determined by (1) incubating thereactive site with a sample containing the analyte for an amount of timethat is sufficient for binding the analyte to the reactive site and (2)measuring an amount of the analyte that binds to the reactive site. Theamount of bound analyte may be measured directly from the reactive siteor indirectly by measuring the depletion of the analyte from the reactedsample, e.g. using the colorimetric Bradford assay or absorbance at 280nm.

In general, the concept of a binding capacity of a bead is explained invarious molecular biology and biochemistry textbooks and is wellunderstood by a skilled person. Some Life Sciences vendors, includingThermoFisher Scientific and New England Biolabs also provide onlineresources that may be used to obtain an estimate of an analyte bindingcapacity for a specific bead. A link to the article titled “Calculatethe Number of Immobilized Proteins per Bead of Agarose AffinitySupports” is provided in the instant specification.

The binding capacity of a bead array for a particular analyte is definedis the maximum amount of the analyte that may specifically bind to thebead array. Bead arrays disclosed in this specification contain reactivesites that have approximately equal binding capacity. Therefore, thebinding capacity of the bead array for a particular analyte isapproximately equal to the binding capacity of a single reactive site,which is capable of binding the analyte, multiplied by the number ofreplicate reactive sites that are present in the bead array. Forexample, the binding capacity of the bead array that contains 5replicate, i.e. identical reactive sites is 5-fold greater than thebinding capacity of one of these reactive sites.

If the target peptide-specific antibody is polyclonal, the reactive siteis potentially capable of binding multiple targets that contain distinctepitopes. Such targets may be derived from a single protein or fromseveral different proteins. The binding capacity of the reactive site inthis case is assumed to be equivalent to the maximum combined amounts ofdistinct analytes, which all contain the epitope that is recognized bythe corresponding antibody.

The methods described in this specification also enable multiplexedquantitative analysis of peptide and/or protein analytes usingsimultaneous affinity capture of distinct analytes on distinct reactivesites of the bead array. While a single reactive site is depicted inFIG. 1A, it is noted that the bead array may contain a distinct reactivesite that contains the reference peptide-specific capture agent 103 anda capture agent that is distinct from the capture agent 104 andrecognizes a target that is distinct from the target 107. The number ofsuch distinct reactive sites in the bead array may be greater than 2,greater than 5, greater than 10, greater than 20, greater than 50 orgreater than 100. This enables development of assays that have amultiplexing capacity that is greater than 2-plex, greater than 5-plex,greater than 10-plex, greater than 20-plex, greater than 50-plex, orgreater than 100-plex.

The bead array may be made to have an analyte binding capacity thatexceeds 1 picomole, 5 picomoles, 10 picomoles, 50 picomoles, or 100picomoles that is sufficient for depleting low, medium and highabundance peptide and/or protein analytes from biological samplescontaining up to 50 milligrams (mg) or more of total input protein. Theanalyte binding capacity of a single reactive site in such bead arraymay exceed 100 femtomoles, 500 femtomoles or 1 picomole.

The duration of the contacting step, i.e. the duration of incubation ofthe bead array with the sample is determined, in part, by the diffusionrate of the analyte within the bead of the reactive site. For beads thatare sufficiently large, e.g. have diameter that exceeds 200 microns(μm), the duration of the contacting step is preferably more than 1hour. In some cases, the contacting step may last more than 3 hours,more than 6 hours or overnight (more than 12 hours). The duration of thecontacting step may be determined by performing a time course study, asdescribed in detail elsewhere in this specification.

When two or more samples are analyzed using the described methods,accurate results will be obtained if it is ensured that the samplescontain approximately equivalent amounts of total input protein. Notethat the above methods do not rely on stable isotope labeling forquantitative analysis of protein abundance changes between the samples.

In an embodiment, the instant specification describes a method fordepleting a peptide analyte from a sample using affinity capture of theanalyte on distinct reactive sites of a bead array, optionally followedby MS detection of the captured analyte from individual reactive sites.The described method is useful for measuring multiple distinct siteswithin a single protein using the methods of bottom-up proteomics.

In reference to FIG. 1B, a sample 126 contains a first peptide 127 and asecond peptide 128 that is distinct from the first peptide. The sampleis brought in contact with a bead array, which contains a first reactivesite 120 and a second reactive site 123. The first reactive sitecontains a bead 121 and a capture agent 122 that specifically recognizesthe first peptide and the second peptide. The second reactive sitecontains a bead 124 and a distinct capture agent 125 that specificallyrecognizes the first peptide and not the second peptide. The bindingcapacity of the bead array may be greater than, equal to, or lower thanan amount of the first peptide in the sample. The binding capacity ofthe bead array may be greater than, equal to, or lower than an amount ofthe second peptide in the sample. Contacting the sample with the beadarray causes the first peptide to bind to the first and the secondreactive sites and the second peptide to bind to the first reactive siteand not the second reactive site. The binding capacity of the bead arraymay be greater than, equal to, or lower than the amount of the firstpeptide 127 in the sample 126. Depending upon the binding capacity ofthe bead array and the duration of the contacting step, the resultingsample 129 may contain an amount of the first peptide, an amount of thesecond peptide, or both. After the contacting step, the reacted reactivesites 129 and 130 are individually analyzed by MS. Analyzing the firstreactive site 129 is used to obtain a ratio of the first peptide to thesecond peptide in the reactive site. The ratio is then used to determinethe amount of the first peptide in the sample. In an embodiment, theamount of the first peptide in the sample is determined quantitatively,e.g. 1.2±0.3 pmol. In an embodiment, the amount of the first peptide inthe sample is determined as being either above or below a specificpre-determined value, such as being greater than 1.2±0.3 pmol, or beinglower than 100±25 fmol.

In an embodiment, the instant specification describes a method forcontacting two or more samples, each of which contains at least twodistinct peptide analytes, with a corresponding number of bead arrays,each of which contains a reactive site recognizing the peptide analytes,optionally followed by MS detection of the captured analytes fromindividual reactive sites. The described method is useful for performingepitope mapping assays.

In reference to FIG. 1C, a first sample 141 contains a first peptide 142and a second peptide 143 that is distinct from the first peptide. Asecond sample 148 also contains the first peptide and the secondpeptide. The first sample is brought in contact with a first bead array,which contains a reactive site 144. The reactive site contains a bead145 and a capture agent 146 that specifically recognizes the first andthe second peptides. A binding capacity of the first bead array isgreater than the amount of the first and the second peptides in thefirst sample. The second sample is brought in contact with a second beadarray, which contains a reactive site 149. The reactive site contains abead 150 and the capture agent 146. A binding capacity of the secondbead array is lower than the amount of the first and the second peptidesin the second sample. The two contacting steps occur concurrently,consecutively or partially overlap in time. After the contacting steps,the reacted reactive site 147 of the first bead array and the reactedreactive site 151 of the second bead array are individually analyzed byMS to obtain a ratio of the first peptide to the second peptide in thecorresponding reactive site.

In an embodiment, the instant specification describes a method forconsecutively contacting a sample that contains at least two distinctpeptide analytes with two or more bead arrays, each of the bead arrayscontaining a reactive site that specifically recognizes the peptideanalytes, optionally followed by MS detection of the captured analytesfrom an individual reactive site of at least one of the bead arrays. Thedescribed method is useful for verifying a quality of the sample,particularly when the analytes in the sample had been subjected to atleast one freeze-thaw cycle.

In reference to FIG. 1D, a sample 160 contains a first peptide 161 and asecond peptide 162 that is distinct from the first peptide. The sampleis brought in contact with a first bead array that contains a reactivesite 163. The reactive site contains a bead 164 and a capture agent 165that specifically recognizes the first and the second peptides. Abinding capacity of the first bead array is greater than, equal to, orlower than the amount of the first and the second peptides in thesample. After the contacting step, the reacted sample 166 now containsreduced amounts of the first and second peptides or be depleted of oneor both peptides. The sample 166 is subsequently brought in contact witha second bead array that contains a reactive site 168. The reactive sitecontains a bead 169 and the capture agent 165. The two contacting stepsmay be separated in time by less than 1 hour, by between 1 hour and 24hours, by between 1 day and 7 days, by between 1 week and 1 month, bybetween 1 month and 12 months, or by more than 1 year. The sample 166may be subjected to 1, 2, 3, 4, 5, or more than 5 freeze-thaw cyclesbetween the two contacting steps. The sample may be stored at roomtemperature, at about 4° C., at about −20° C., at about −80° C. or below−80° C. between the two contacting steps. The reacted reactive site 167of the first bead array and/or the reacted reactive site 170 of thesecond bead array are optionally analyzed by mass spectrometry to obtaina ratio of the first peptide to the second peptide in the correspondingreactive site. In an embodiment, the peptides that are depicted in FIG.1A, FIG. 1B, FIG. 1C or FIG. 1D and described in the correspondingsections of the specification are unmodified peptides that do notcontain a chemical tag. Specifically, they do not contain a chemicalgroup, which is a constituent of one of the labeling reagents known asisotope-coded affinity tag (ICAT®), mass differential tags for relativeand absolute quantification (mTRAQ®), isobaric tags for relative andabsolute quantitation (iTRAQ®) and TMT™.

After performing the contacting step that is depicted in any of FIGS. 1Athrough 1D, the reacted bead array(s) is prepared for MS analysis. Thisincludes removing non-specifically bound compounds by washing thereactive sites with a liquid medium that contains a mild detergent, abiological buffer or just deionized water. The washed bead array is thenplaced on a specially designed plate, which contains an array ofmicrowells that are dimensioned to accept no more than one reactive siteper microwell. Placing the bead array on the microwell array platecauses individual reactive sites to sink into individual microwells,thereby creating a planar bead array. Analytes captured on individualreactive sites are released into the respective microwells and mixedwith a matrix for MS. For increased efficiency of the MS measurement,the microwell array plate may be converted into a flat surface slidethat contains a surface array of spots containing analytes eluted fromindividual reactive sites. The array of spots is measured by MALDI MS oranother type of MS. Some of the array processing steps described in thisparagraph are explained in greater detail below.

The microwell array plate is made of a silicone gasket containing anarray of through holes that is reversibly attached to a flat surface ofa microscope slide. The slide surface is electrically conductive, forexample it may contain a surface layer of Indium Tin Oxide (ITO), goldor another conductive material. The watertight seal formed by thesilicone gasket in contact with the slide surface prevents liquiddiffusion and/or leakage from individual wells, both for aqueous andorganic liquids, such as acetonitrile.

The microwell array plate may be subdivided into several regions, e.g.2, 4, 8, 16, 24, 64 regions using a multi-chamber frame. The individualregions of the microwell array plate may contain beads with analytesderived from distinct biological samples. The multi-chamber frame isremovably attached to the microwell array plate using plastic orstainless steel clips.

In an embodiment, the specification describes a reusable microarraysubstrate for eluting analytes from bead arrays. The reusable microarraysubstrate includes an elastomer member, i.e. an elastomer gasket that isremovably bonded to a flat surface of a solid support, e.g. a microscopeslide. The elastomer gasket contains an array of through holes and avisual marking for identifying a surface of the elastomer gasket that isconfigured for bonding to the solid support. In an embodiment, adiameter of a through hole and a distance between adjacent through holesare less than 600 microns.

The suitable slide is an ITO or gold-coated 25×75×1.1 mm microscopeslide that is available from multiple vendors. An exemplary elastomer(silicone) gasket containing an array of through holes and methods ofattaching the gasket to the slide to make the microarray substrate aredescribed in the U.S. patent application Ser. No. 16/125,164,publication No. US 2019-0072546 A1. However, the above reference doesnot teach that the individual components of the microarray substrate maybe reusable, that is, may be used multiple times for eluting analytesfrom bead arrays. Reusing the slide is relatively straightforward andrequires a sufficiently thorough wash to remove traces of previouslyeluted analytes and the matrix from the slide surface. This may beachieved by using a detergent followed by rinsing the slide severaltimes with deionized water. On the other hand, the chemical propertiesof the elastomer gasket may change significantly between a first use andsubsequent uses due to exposure of the elastomer surface to the harshreagents in the matrix solution, such as strong acid and organicsolvent. Importantly, as these reagents may irreversibly change theadhesive properties of the elastomer, they will prevent effectivebonding between the slide and the gasket. To overcome this problem, asurface of the elastomer gasket that has been previously exposed to theMALDI matrix solution (the “top” surface) needs to be distinguished froman opposite surface that was in contact with the slide (the “bottom”surface) and therefore has not been exposed to the reagents of thematrix solution, as these surfaces may have distinct adhesiveproperties. This is achieved by including a visual marking in the gasketthat enables unambiguous identification of the surface that is suitablefor bonding to the solid support. The visual marking may be provided inthe form of a chamfer so that the gasket is asymmetrically shaped.

In an embodiment, an elastomer (e.g. silicone) gasket is originally madesuch that its “top” surface has different adhesive properties than its“bottom” surface. The top surface is the surface that is facing up whenthe gasket is attached to a solid support (the microscope slide) and isalso the surface that temporarily contacts the multi-chamber frame thatis used to sub-divide the microwell array. The bottom surface is thesurface that is facing down, contacts the solid support (the microscopeslide) and forms a fluidic seal that fluidically separates individualmicrowells (through holes that are fluidically sealed at the bottom bythe solid support). While both the top and the bottom surfaces may bemade of a same material, their adhesive properties may be designed to besignificantly different. Specifically, the bottom surface may havebetter self-adhesive properties compared to the top surface, such thatthe bottom surface readily forms a fluidic seal between microwells(through holes) when attached to a flat surface solid support, while thetop surface does not readily form a fluidic seal.

The top surface of a silicone gasket may be made less adhesive than theopposite bottom surface by intentionally increasing its surfaceroughness. A greater roughness may be achieved by allowing burrs to formaround openings into through-holes on the top surface of the gasket,while preventing burrs from forming on the bottom surface. A burr iscommonly defined as a raised edge of material that remains attached to aworkpiece after a modification process. Methods of both creating andpreventing burrs while laser cutting of through holes in siliconeelastomer sheets, which are less than 1 mm thick, are well known in theindustry. A manufacturer of silicone gaskets such as Grace Bio-Labs(Bend, Oreg.) may be requested to render one surface of the gasket lessadhesive than the opposite surface by intentionally allowing burrs toform on the former. Since burrs are only formed around openings intothrough holes (microwells), an area of the top surface that is locatedbetween peripheral through holes and the edges of the gasket remainsfree of burrs and is therefore capable of forming a fluidic seal whenattached to a multi-chamber frame. An aqueous medium placed inside amulti-chamber frame attached to a microwell array plate containing burrswill freely travel between adjacent chambers while micron-sized beadswill remain localized within their respective chambers due to sizeconstrains. It is estimated that dimensions of burrs produced by lasercutting in silicone sheets do not exceed 5 microns and often do notexceed 1 micron.

Therefore, the specification describes a microwell array plate thatincludes a flat surface solid support (e.g. a microscope slide) and aremovably bonded elastomer member (e.g. a silicone gasket), in which theelastomer member has a first (bottom) surface and an opposite second(top) surface. The bottom surface is essentially free of burrs andcapable of forming a fluidic seal with the solid support. The topsurface includes an area that contains burrs around openings intomicrowells and also includes an area that is essentially free of burrs,the latter area being located between peripheral microwells and theedges of the gasket. The burr-free area is preferably less than 50% of atotal area of the top surface and capable of forming a reversiblefluidic seal with a multi-chamber frame. The elastomer member furtherincludes a chamfer or a similarly functioning visual marking thatenables the burr-free surface to be distinguished from theburr-containing surface.

While regular grade polydimethylsiloxane (PDMS, silicone) is well suitedfor elastomer gaskets, certain improvements in the gasket performancemay be achieved by using fluorocarbon based synthetic rubbers, such asfluoroelastomer FKM, e.g. VITON®, perfluoroelastomer FFKM, or FEPM(tetrafluoroethylene propylene), e.g. AFLAS®. Fluoro-rubbers such asFKM, FFKM and FEPM have good adhesive properties, yet possess greatermechanical sturdiness, which may be beneficial for maintaining regulargrid of the microarray spots.

In an embodiment, the instant specification describes a microarraysubstrate that is coated with a layer of crystalline MALDI matrix. Onebenefit of using the substrate that is pre-coated with the matrix isthat the bead-eluted analytes may better incorporate into the matrixlayer that is already positioned on the bottom of individual microwellsthereby generating stronger signal from the eluted analytes. Unlike theexisting substrates containing pre-spotted matrix, the matrix-coatedmicroarray substrate should have a near neutral pH and not contain astrong acid because low pH may cause premature dissociation of analytesfrom beads before the beads are positioned into individual microwells.In an embodiment, a near neutral pH is defined as being greater than5.0±0.5 and lower than 9.0±0.5 pH units. In an embodiment, a nearneutral pH is defined as being greater than 6.0±0.5 and lower than8.0±0.5 pH units.

Another benefit of using the microarray substrate that contains apre-spotted layer of MALDI matrix is that such microarray substrate isable to more efficiently retain an aqueous medium, e.g. deionized waterinside individual microwells and therefore extend an amount of time,during which beads that are placed inside individual microwells remainhydrated. While microwell structures, which are made of silicone, glass,ITO or metals, do not efficiently absorb and retain water, microwellstructures, which are made of these materials and additionally contain asurface layer of MALDI matrix, such as α-cyano-4-hydroxycinnamic acid(CHCA) and sinapinic acid (SA), are able to absorb and retain waterand/or aqueous solutions for an extended amount of time, e.g. greaterthan 5 minutes, greater than 10 minutes or greater than 15 minutes underconditions of ambient humidity. The surface layer of MALDI matrix coversboth bottom surfaces and sidewalls of individual microwells, as well asthe area between openings into individual microwells. In an embodiment,the ambient humidity is higher than 15% relative humidity at atemperature 15° C. or higher.

In an embodiment, the specification describes a method for eluting oneor more analytes from a bead array. The described method includes thesteps of receiving a bead array, which contains an analyte-bound beadlocated inside a microwell filled with liquid aqueous medium, andrepeatedly or continuously depositing liquid elution medium into themicrowell, the elution medium containing a dissolved matrix for massspectrometry and a solvent, such that a rate, at which the elutionmedium is being deposited into the microwell, is approximatelyequivalent to a rate, at which the solvent is escaping from themicrowell via evaporation. The bead remains continuously exposed to thesolvent and a solution that forms in the microwell has a higherconcentration of the dissolved matrix relative to the original elutionmedium.

While earlier references describe applying MALDI matrix solutions tobead arrays, they generally teach using saturated solutions of matrix,such as 10 mg/ml of CHCA and drying bead arrays between consecutivecycles of matrix application. These methods may not achieve optimalelution of bead-bound analytes in cases where beads are composed ofporous materials such as agarose or cellulose.

An improved method of analyte elution, which is described here, involvesusing a more dilute MALDI matrix solution, e.g. 5 mg/ml of CHCA,applying the matrix solution to a microwell that contains a sufficientamount of aqueous medium, for example deionized water, and matching arate of depositing the matrix solution into the microwell to a rate ofevaporation of a solvent of the matrix solution from the microwell sothat the composition of the medium inside the microwell graduallychanges from neutral, aqueous medium to acidic, organic-solventcontaining medium, which causes the affinity-bound analytes todissociate from their respective beads and remain in a solution for aspecific amount to achieve efficient incorporation of the analytes intothe MALDI matrix. The described conditions also prevent splattering andspilling of liquids from the microwells thereby minimizing ofeliminating potential cross-talk between neighboring microwells.

Exemplary bead arrays and methods of making bead arrays are described inthe U.S. patent application Ser. No. 16/125,164, publication No. US2019-0072546 A1. The above reference enables making of a bead array thatcontains affinity beads with bound peptide analytes located insideindividual microwells of a microwell array that consists of a removablesilicone gasket attached to an ITO-coated glass microscope slide.Individual microwells are filled with deionized water.

The matrix solution for eluting analytes from a bead array should besufficiently dilute. For the common CHCA matrix, the concentrationshould be less than 10 mg/ml; specifically, 6 mg/ml or less, 4 mg/ml orless, 2 mg/ml or less, or 1 mg/ml or less. The matrix solution shouldalso contain a sufficiently high concentration of acid; specifically,more than 0.2%, more than 0.3% or more than 0.4% (v/v) of eithertrifluoroacetic acid (TFA), formic acid (FA) or other suitable acid suchas hydrochloric acid. The matrix solution should also contain asufficiently high concentration of an organic solvent, e.g. at least30%, at least 40% or at least 50% (v/v) of either acetonitrile, ethanol,isopropanol or methanol. Because the initial concentration of the matrixin the elution medium is sufficiently low, the dilution of the organicsolvent, which occurs when the matrix solution-containing aerosoldroplets enter water-filled microwells, does not cause immediatecrystallization and precipitation of the CHCA matrix yet the pH of theresulting mixed solution that forms in the microwells is sufficientlylow, e.g. below 2 due to the high acid content of the matrix solution,which causes rapid acidification and dissociation of the affinitycaptured peptides from antibody-conjugated beads and subsequent mixingof the eluted peptides with the matrix-containing solution inside themicrowells.

The elution medium, i.e. the matrix solution is being continuouslydeposited into the microwells via repeated cycles of applying an aerosolcontaining microdroplets of the matrix solution into microwells. Thatprocess occurs simultaneously with a process of continuous evaporationof the solvent of the matrix solution from the microwells therebycausing an increase in the concentration of the dissolved matrix in themicrowells to the point where the matrix concentration in the mixture ofthe aqueous medium and the elution medium eventually exceeds the matrixconcentration in the original elution medium. Once the matrix depositionprocess stops, there is provided a sufficient time for the solvent toevaporate, which causes the matrix to precipitate and co-crystallizewith the eluted peptide analytes. Because the analyte elution from beadsoccurs before the matrix crystallization, the eluted peptides areprovided a sufficient amount of time to thoroughly mix with thedissolved matrix, e.g. for more than 5 minutes, more than 10 minutes,more than 20 minutes, or more than 30 minutes. If desired, the matrixcrystallization process may be visually monitored using opticallytransparent ITO-coated glass slides.

The deposition of the elution medium into the bead array should beperformed using conditions that prevent splattering and spilling of themedium from the microwells in order to maintain the spatial resolutionof the array and to prevent cross-talk between adjacent spots within thearray. Specifically, the microdroplets containing the elution mediumshould have sufficiently low velocity to minimize their impact uponcontact with the liquid medium inside the microwells, which may beachieved by selecting sufficiently low flow rate of the carrier gas thatis used to generate the aerosol. Other conditions may include selectingan optimal distance between the nozzle that generates the microdropletsand the surface of the array. In an embodiment, the distance is greaterthan 30 mm, greater than 45 mm or greater than 60 mm.

Furthermore, if the aerosol containing the elution medium is generatedusing a programmable device, e.g. a MALDI matrix sprayer, values of theparameters that are supplied to the device, such as the speed of matrixdeposition and the density of matrix solution per area unit should beselected according to an ambient air humidity level. When the values areproperly selected, the microwells do not overflow because the additionof elution medium into the microwells is matched by the escape of thesolvent of the elution medium from the microwell via evaporation. Forthe user convenience, the programmable device may include a hygrometerthat is operably connected to the device.

The values of parameters provided to the programmable device may befurther selected according to the chemical composition of the analyte,the chemical composition of the elution medium and/or the chemicalcomposition of the aqueous medium.

Once the matrix solution application stops, the residual solvent isallowed to completely evaporate, which causes the matrix to precipitateand co-crystallize with the eluted peptide analytes. The beads becomedry and may be removed from their respective microwells by compressedair, for example using a regular duster can. The bead removal shouldtake place prior to separating the gasket from the slide, althoughapplying the air to dislodge beads from the slide after the gasket hadbeen detached is also possible.

The methods described above enable fabrication of a microarray thatcontains multiple discrete spots located on a flat surface of a solidsupport, e.g. a microscope slide. Such microarray is schematicallydepicted in FIG. 2A. All microarray spots contain MS matrix and at leastsome spots additionally contain one or multiple analytes transferredfrom a bead array. A spot that contains or is suspected of containing ananalyte is visibly distinct from a spot that is devoid of the analyte.The analyte-containing spot is also distinct from a spot that containsor is suspected of containing a mixture of analytes, as explained ingreater detail below. The microarray may contain a smaller size section201 that contains analytes obtained from a single biological sample. Themicroarray may contain distinct sections that contain analytes obtainedfrom distinct biological samples. The microarray may further contain afiducial 202, as described in greater detail below.

In reference to FIG. 2B, when a bead occupies a certain portion of abottom surface within a microwell, it may prevent the matrix fromprecipitating and forming crystals directly underneath. After the beadis removed, the pattern of matrix distribution within each spot isrevealed. A spot 203 that previously contained a bead exhibits acharacteristic donut or crescent shape because it includes an innerregion 204 that has a visibly lower density of matrix coverage; in somecases, the inner region 204 may be substantially free of the matrix. Anarea of such inner region may be greater than 5%, greater than 10%,greater than 20%, greater than 30%, greater than 40%, greater than 50%,greater than 60%, greater than 70%, greater than 80%, or greater than90% of a total area of the spot, being determined by a combination ofseveral factors: the bead diameter, the microwell diameter, surfaceproperties of the microarray slide, chemical composition of the matrixsolution and conditions of the matrix crystallization process. Bycontract, a spot 205 that did not initially contain a bead exhibits asubstantially uniform pattern of the matrix coverage compared to theformer spot. The distinct shapes of the two types of spots are visuallydistinguishable and may be used to selectively measure spots thatcontain or are suspected of containing an analyte of interest whileexcluding spots that are not expected not contain such analyte.

Furthermore, an analyte-containing spot 203 is also visibly distinctfrom spots 206 and 207 that contain or are suspected of containing amixture of analytes. The mixing of analytes may occur if two, three ormore beads occupy a single microwell. In such case, a pattern of matrixdistribution within a spot reveals two or three distinct inner regionsthat have a visibly lower density of matrix coverage or are free of thematrix. The distinct shape of a spot that previously contained multiplebeads and therefore contains or is suspected of containing a mixture ofanalytes may be used to exclude such spot from analysis by MS.Alternatively, it may be used in data analysis to indicate that multipleanalytes were co-eluted from several beads in a particular spot.

Due to inherent heterogeneity of the matrix-analyte crystallizationprocess, the spatial distribution of an analyte within ananalyte-containing spot may not be known prior to MS analysis,particularly if the analyte is not fluorescent. However, it is notedthat spots, which are prepared using the above-described method andcontain an analyte, consist of an upper layer and a lower layer, bothlayers containing the matrix yet the upper layer containing a greateramount of the analyte compared to the lower layer. The higher analyteconcentration in the upper layer of a spot may be experimentallyverified by continuously acquiring MS data from the spot and detecting adecrease in intensity of the analyte signal after a top layer of thespot has been consumed, in some cases without visibly depleting thematrix in the spot. Thus, it is possible to limit the data acquisitionto the upper layer of a particular spot to reduce the amount of timeneeded to interrogate the spot.

In addition to the analyte-devoid spots that are randomly positionedthroughout the microarray, the microarray may contain at least one rowand/or column that consists of spots 208 that are devoid of bead-elutedanalytes and have a visibly distinct pattern of the matrix coverage.Such row and/or column(s) is located on a periphery of the microarray orat a boundary 209 that separates sections of the microarray that containanalytes obtained from distinct samples. Its location coincides with alocation of a divider of a multi-well chamber. As the multi-well chamberis pressed against the elastomer gasket during the sample preparationprocess, through-holes of the gasket that are located directly under thedivider do not receive water or beads. Accordingly, when the bead arrayassembled on a microwell array plate is subsequently contacted with thematrix-containing aerosol, at least one row and/or column contains wellsthat are initially dry and do not contain beads. The pattern of matrixcoverage in such wells is noticeably different from the wells thatcontained water, even if both types of wells do not contain a bead. Thelocation of an empty row and/or column is determined by the layout ofthe multi-well chamber and may be further verified by visual detection.During MS data acquisition process, spots within such row and/or columnmay be excluded from the measurement to provide significant timesavings. In the context of current specification, a row or a column of amicroarray is defined as a group that consist of at least 3 microarrayspots that are aligned horizontally or vertically, respectively.

Furthermore, the molecular structure of matrix crystals formed inlocations that were initially dry is different from the molecularstructure of matrix crystals in locations that initially contained theaqueous medium resulting in noticeable differences in the correspondingmass spectra. Specifically, a mass spectrum recorded from a spot 208that is produced by placing the MS matrix solution into a dry microwellexhibits multiple strong peaks in the spectral region below 1400 m/z,which are assigned to various matrix clusters containing sodium andpotassium ions. By contrast, a mass spectrum recorded from a spot 205that is produced by placing the matrix solution into a water-filledmicrowell exhibits much weaker matrix clusters peaks that are often notdetectable above 800 m/z. Therefore, in applications where multiplespots of a microarray are analyzed by MS, the location of a row and/orcolumn that separates sections of the microarray containing analytesfrom different biological samples may be determined by detecting intensematrix cluster signals from individual spots within such row and/orcolumn. The described procedure may be used for microarray gridding.

The asymmetrical shape of the elastomer gasket containing a chamferallows placing a fiducial marker into the microarray during the processof matrix application. The fiducial 202 contains the matrix and has ashape that is visibly distinct from a shape of a microarray spot. Aposition of the fiducial coincides with a location of the chamfer. Thefiducial is produced at the same time as the analytes are being elutedfrom the bead array during the matrix application process. Thus, thequality of matrix crystals in the fiducial should be essentiallyidentical to the quality of matrix crystals in the individual microarrayspots and may be used to monitor the conditions of the matrixapplication. Once created, the fiducial may be used to properly orientthe microarray before placing the microarray slide into an MSinstrument. Alternatively, the fiducial may be measured during MSimaging of the microarray and its position used to properly orient themicroarray image for subsequent data analysis.

Individual spots of the described microarrays may be 1000 microns orless, 600 microns or less, 400 microns or less, or 200 microns or less.

Distinct patterns of analyte-containing spots (203) and analyte-devoidspots (205) within each microarray are due to the random nature of aprocess of beads being placed inside microwells, the number of availablemicrowells being greater than the number of beads. Such distinctpatterns of analyte-containing spots and/or analyte-devoid spots may beused for positional encoding of individual microarrays. Specifically,X-Y coordinates of either all or some of analyte-containing spots and/orall or some of analyte-devoid spots in a microarray may be associatedwith various information about the microarray, including for exampleidentities of analytes present in the microarray, a date of making themicroarray, etc. Alternatively, an optical image of the microarrayrather than X-Y coordinates may be associated with the information aboutthe microarray. The concept of positional encoding of a microarray byusing the unique patterns of analyte-containing and analyte-devoid spotsis also described in Experimental Examples.

In summary, the instant specification describes several improvementsrelated to making, using and analyzing bead arrays by MS.

In an embodiment, the specification describes a microarray substratecomprising an elastomer member that is removably bonded to asubstantially flat surface of a solid support, the elastomer membercontaining a plurality of through holes and a visual marking, wherein adiameter of a through hole and a distance between adjacent through holesare less than 600 microns and the visual marking is suitable foridentifying a surface of the elastomer member that is configured forbonding to the solid support. In an embodiment, the elastomer member isasymmetrically shaped. In an embodiment, the elastomer is afluoro-elastomer. In an embodiment, the surface configured for bondingto the solid support and an opposite surface of the elastomer memberhave distinct adhesive properties. In an embodiment, portions of theelastomer member and the solid support are coated with a surface layerthat contains a crystalline matrix for MS and does not contain a strongacid.

In an embodiment, the specification describes a method for making areusable microarray substrate, the method comprising the step of bondingan elastomer member to a substantially flat surface of a solid support,the elastomer member containing a plurality of through holes and avisual marking, wherein a diameter of a through hole and a distancebetween adjacent through holes are less than 600 microns, and the visualmarking is used to orient the elastomer member relative to the solidsupport prior to the bonding step. In an embodiment, the visual markingis suitable for identification of a surface of the elastomer member thatwas not previously exposed to a solution containing a matrix for MS. Inan embodiment, the method further comprises the step of contacting theelastomer member and the solid support with an aerosol, the aerosolcontaining a dissolved matrix for MS and not containing a strong acid,the contacting step being performed after the bonding step.

In an embodiment, the specification describes a microarray comprising aplurality of discrete spots positioned on a substantially flat surfaceof a solid support wherein a spot that contains or is suspected ofcontaining an analyte is visibly distinct from a spot that is devoid ofthe analyte and all spots contain a matrix for MS. In an embodiment, theanalyte-containing spot includes an inner region that has a visiblylower density of the matrix coverage. In an embodiment, an area of theinner region is greater than 20% of a total area of theanalyte-containing spot. In an embodiment, the analyte-containing spotis further visibly distinct from a spot that contains or is suspected ofcontaining a mixture of analytes. In an embodiment, theanalyte-containing spot comprises an upper layer and a lower layer, bothlayers containing the matrix, the upper layer containing a greateramount of the analyte compared to the lower layer. In an embodiment, themicroarray contains at least one row and/or column that consists ofspots that have a visibly distinct pattern of the matrix coverage, theat least one row and/or column being located on a periphery of themicroarray or at a boundary that separates sections of the microarraythat contain analytes obtained from distinct samples. In an embodiment,the microarray further comprises a fiducial, the fiducial containing thematrix and having a shape that is visibly distinct from a shape of amicroarray spot. In an embodiment, dimensions of individual microarrayspots are 400 microns or less.

In an embodiment, the specification describes an analytical method, themethod comprising the steps of receiving a microarray, the microarraycomprising a plurality of discrete spots positioned on a substantiallyflat surface of a solid support, all spots containing a matrix for MS,optically identifying a spot that includes an inner region that has avisibly lower density of the matrix coverage, optically identifying aspot that has a substantially uniform pattern of the matrix coverage,analyzing the former spot by MS, and excluding the latter spot fromanalysis by MS. In an embodiment, the excluded spot is located on aperiphery of the microarray or at a boundary that separates sections ofthe microarray that contain analytes obtained from distinct samples. Inan embodiment, the method further comprises the step of opticallyidentifying and subsequently excluding multiple spots from the analysisby MS, the multiple spots forming at least one row and/or column withinthe microarray, the at least one row and/or column being located on aperiphery of the microarray or at a boundary that separates sections ofthe microarray that contain analytes obtained from distinct samples.

In an embodiment, the specification describes a method for eluting ananalyte from a bead array, the method comprising the steps of receivinga bead array, the bead array comprising a bead that is positioned in amicrowell, the bead being associated with an analyte, the microwellcontaining an aqueous medium, and repeatedly depositing an elutionmedium into the microwell, the elution medium comprising a dissolvedmatrix for MS and a solvent, wherein a rate, at which the elution mediumis being deposited into the microwell, is approximately equivalent to arate, at which the solvent is escaping from the microwell viaevaporation, such that: the bead is continuously exposed to the solventand a solution that forms in the microwell has a higher concentration ofthe dissolved matrix relative to the elution medium. In an embodiment, aconcentration of the matrix in the elution medium is less than 10 mg/ml.In an embodiment, the solvent contains more than 0.2% of a strong acid.In an embodiment, the bead is continuously exposed to the elution mediumfor at least 5 minutes. In an embodiment, elution medium is beingdeposited into the microwell using conditions that substantially preventsplattering and spilling of the elution medium from the microwell. In anembodiment, the elution medium is being deposited using a programmabledevice that is capable of producing an aerosol, the method furthercomprising the step of supplying at least one value to the programmabledevice, the at least one value being selected according to an ambientair humidity level. In an embodiment, the at least one value is furtherselected according to at least one of a chemical composition of theanalyte, a chemical composition of the elution medium, and a chemicalcomposition of the aqueous medium. In an embodiment, the programmabledevice is operably connected to a hygrometer.

In an embodiment, the specification describes a method for eluting ananalyte from a bead array, the method comprising the steps of receivinga bead array, the bead array comprising a bead that is positioned in amicrowell, the bead being associated with an analyte via an acid-labilebond, the microwell containing an aqueous medium, depositing a firstamount of an elution medium into the microwell, the elution mediumcomprising a dissolved matrix for MS and a solvent, and allowing theaqueous medium and the elution medium to form a mixture, the mixturebeing sufficiently acidic to cause the analyte to dissociate from thebead, depositing a second amount of the elution medium into themicrowell such that a concentration of the matrix in the mixture exceedsa concentration of the matrix in the elution medium and allowing thesolvent to evaporate from the microwell thereby causing the matrix toco-crystallize with the analyte.

In an embodiment, the specification describes a method for eluting ananalyte from a bead array, the method comprising the steps of receivinga bead array, the bead array comprising a bead that is positioned in amicrowell, the bead being associated with an analyte, the microwellcontaining an aqueous medium, depositing an elution medium into themicrowell, the elution medium comprising a dissolved matrix for MS and asolvent, wherein the aqueous medium and the elution medium are allowedto form a mixture under conditions that substantially prevent the matrixfrom precipitating from the mixture, and providing a sufficient amountof time to allow the solvent to escape the microwell via evaporationthereby causing the dissolved matrix to precipitate. In an embodiment,the amount of time is greater than 10 minutes.

The present disclosure is described in the following Examples, which areset forth to aid in the understanding of the disclosure, and should notbe construed to limit in any way the scope of the disclosure as definedin the claims which follow thereafter. The following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the presentdisclosure, and are not intended to limit the scope of the presentdisclosure nor are they intended to represent that the experiments beloware all or the only experiments performed. Efforts have been made toensure accuracy with respect to numbers used (e.g. amounts, temperature,volume, time etc.) but some experimental errors and deviations should beaccounted for.

EXAMPLES Materials and Equipment

N-Hydroxysuccinimide (NHS)-activated magnetic agarose beads, ITO (IndiumTin Oxide) and gold-coated microscope slides, silicone gaskets andmulti-well chambers and methods of assembling bead arrays are describedin the U.S. patent application Ser. No. 16/125,164, publication No. US2019-0072546 A1.

Unless noted otherwise, consumables such as microcentrifuge tubes,pipette tips, weigh boats etc., were standard research grade. Reagentssuch as organic solvents, acids, salts, buffers, detergents, MALDImatrices etc., were standard research grade with a purity of 99% orhigher and used as received from the manufacturer without furtherpurification. Standard lab equipment included a microcentrifuge, amicroplate centrifuge, magnetic tube racks, microtiter plate shaker,vortexer, etc.

Programmable robotic liquid sprayer iMatrixSpray that is capable ofdispensing MALDI matrix solutions was from Tardo GmbH (Subingen,Switzerland).

EXPERIMENTAL RESULTS

Some of the experiments performed using the compositions and methodsdisclosed in this application and the resulting experimental data aredescribed below.

Example 1 Microarray Reactive Site Containing Two Distinct CaptureAgents

Rabbit monoclonal antibody recognizing p44 and p42 MAP Kinase (Erk1 andErk2) when dually phosphorylated at Thr202 and Tyr204 of Erk1 (Thr185and Tyr187 of Erk2), and/or singly phosphorylated at Thr202 waspurchased from Cell Signaling Technology (Danvers Mass.), catalog number4370 (D13.14.4E). Rabbit monoclonal antibody recognizing phosphorylatedThr1462 in human Tuberin (TSC2) was also from Cell Signaling Technology,catalog number 3617 (5B12). The #3617 and #4370 antibodies were suppliedby the manufacturer in BSA-free, glycerol-free and Tris-free medium.After receiving the antibody stocks, their concentrations were adjustedto 1 mg/ml by diluting with 1×PBS. An antibody mixture was prepared bycombining stocks containing the #4370 and #3617 antibodies in 95:5 ratio(w/w). An amount of approximately 7 micrograms of the combined antibodymixture was used for direct conjugation to 100 of Protein A+G activatedmagnetic agarose beads. Individual beads were in the 375 to 400 microndiameter range. The beads containing the mixture of two antibodies weresubsequently cross-linked to the beads using the previously publishedprocedure utilizing dimethyl pimelimidate dihydrochloride (DMP). Anamount of capture reagent per single bead was estimated to be between400-500 fmol and 20-25 fmol for the #4370 and #3617 antibodies,respectively.

The beads were subsequently transferred into 1×PBS supplemented with0.03% sodium azide and stored at 4° C. until ready to use.

Example 2 Measuring the Analyte Binding Capacity of a Bead Array

Multiple identical reactive sites containing the antibodies #3617 and#4370 were prepared as described in the previous Example. Severalidentical bead arrays were assembled by combining 5 replicate reactivesites per bead array. The binding capacity of each bead array is assumedto be 5-fold greater than the binding capacity of a single reactivesite. It was experimentally determined that the #4370 antibody iscapable of specifically binding several peptide targets containing therecognized epitope, including peptides VADPDHDHTGFL[pT]EYVATR (SEQ IDNO: 1) (fragment of Erk2, singly phosphorylated at Thr185),VADPDHDHTGFL[pT]E[pY]VATR (SEQ ID NO: 2) (fragment of Erk2, doublyphosphorylated at Thr185 and Tyr187), IADPEHDHTGFL[pT]EYVATR (SEQ ID NO:3) (fragment of Erk1, singly phosphorylated at Thr 202) andIADPEHDHTGFL[pT]E[pY]VATR (SEQ ID NO: 4) (fragment of Erk1, doublyphosphorylated at Thr 202 and Tyr204). The above peptides are derivedfrom their respective precursor proteins via proteolytic digestion usingtrypsin. In addition, it was experimentally determined that the #4370antibody is capable of specifically binding various synthetic peptidescontaining the recognized epitope, including peptides that are bothlarger and smaller than the tryptic fragments of Erk1 and Erk2.

The antibody #3617 specifically recognizes phosphorylated Thr1462 infull-length human Tuberin. It was experimentally determined that the#3617 antibody is not cross-reactive and does not specifically recognizeendogenous peptides that are present in digested mammalian cell lysates,human or mouse tissue lysates or biofluids such as serum and plasma. Itwas also determined that the #3617 antibody does not specificallyrecognize the corresponding proteolytic fragment of human Tuberin afterdigestion with trypsin, likely due to the loss of the epitope. Asynthetic peptide GSPSGLRPRGY[pT]ISDSAPSR (SEQ ID NO: 5) where [pT]denotes phosphorylated threonine was synthesized by New England Peptide(Gardner, Mass.) and supplied in greater than 95% purity. The peptideamount was quantified using amino acid analysis (AAA). The syntheticpeptide contains the phospho-Tuberin epitope that is recognized by the#3617 antibody. The synthetic peptide is not tryptic as it contains anadditional amino acid, the N-terminal Gly that is not found in theprecursor human protein and also contains a missed trypsin cleavagesite. Accordingly, when the synthetic peptide is spiked into a sample,the sample will contain more than 95% of a single peptide, i.e. thesynthetic peptide that is specifically recognized by the #3617 antibodyand less than 10%, in fact less than 5% of other peptides that arerecognized by the #3617 antibody, the other peptides being impurities ofthe synthetic peptide. The synthetic peptide is detected by MALDI TOF MSnear 2141.0 m/z (singly charged ions) and 1071.0 m/z (doubly chargedions). Minimizing the presence of additional peptides in the sample tobelow 5% enables acquisition of very clean MS spectra that exhibit asingle strong peak at the m/z of singly charged ion and a smaller peakat the m/z of doubly charged ion.

The binding capacity of the assembled bead array was evaluated inseveral experiments. Measured amounts (1 pmol to 10 pmol) of thesynthetic peptide described in the previous paragraph were mixed with asolution containing approximately 50 pmoles of trypsin-digested BovineSerum Albumin (BSA) and incubated with the bead arrays. The bead arrayswere incubated with the peptide-containing solution for at least 1 hourand at most 24 hours. The analyte signal from individual reactive sitesbeads was measured by MS as described elsewhere in the specification.The amount of residual synthetic peptide in a particular sample after anincubation with a bead array was measured using the #3617 antibody andthe conventional dot blot assay. In some experiments, the reactedsolution was saved and subsequently incubated with a new bead array.

It was experimentally determined that a bead array containing 5identical reactive sites, each of the reactive sites containing 5% ofthe #3617 antibody, has a binding capacity that is less than 15% of 1pmol of the corresponding synthetic peptide. The estimate is derivedfrom both the MS data, which measures the bound peptide, and the dotblot data, which measures the peptide remaining in the sample after theincubation. The estimate is also in agreement with the calculated amountof the antibody in the bead array. The results indicate that a beadarray containing 100 reactive sites has a binding capacity that is lessthan 50% of 10 pmol of the #3617-specific synthetic peptide. For greatermultiplex assays, the amount of synthetic peptide in a sample may beincreased to more than 50 pmol or even more than 100 pmol.

The binding capacity of the same bead array for the Erk1/2-derivedpeptides is significantly higher due to a greater amount of the #4370antibody compared to the #3617 antibody. It is estimated that the beadarray is capable of binding at least 2.5 pmol of a combined amount ofpeptides that contain the epitope recognized by the former antibody.That includes both singly- and doubly-phosphorylated fragments of Erk1and Erk2.

Example 3 Preparing a Biological Sample for MS Analysis

The procedures for culturing human MKN45 cells, lysing the cells,digesting the cell lysates and purifying the proteolytic peptides areprovided in the U.S. patent application Ser. No. 16/125,164, publicationNo. US 2019-0072546 A1. The procedure for obtaining proteolytic peptidesfrom a human tissue (adult brain) is provided below.

A total of 100 mg of adult human male brain tissue was received from theMaine Medical Center BioBank (Scarborough Me.). The tissue sample wascooled in liquid nitrogen for 15 minutes and subsequently pulverizedusing the Bessman tissue pulverizer. The pulverized material wastransferred to a clean polypropylene tube, the urea lysis buffer wasadded to the sample to bring the total protein concentration to 2 mg/mL.In some cases, further homogenization was performed using a bead beaterand 0.1 mm zirconia/silica beads (BioSpec Products, catalog number11079101z). The complete the tissue lysis, the mixture was sonicatedusing a microtip at 15 W output with 3 bursts of 15 sec each withcooling on ice for 1 min between each burst. The lysate was cleared bycentrifugation at 4,800 g for 15 min at 15° C. and the supernatantcontaining the protein extract transferred into a new tube. At thisstage the tissue lysate was frozen at −80° C. and stored for severalweeks. Protein denaturation, reduction, alkylation, desalting andlyophilization procedures for tissue-derived proteins were identical tothe previously described procedures for cell culture-derived proteins.The lyophilized peptides can be stored frozen at −80° C. for severalmonths.

Example 4 Obtaining an Estimate of the Total Protein Content of a Sample

A lyophilized sample containing peptides obtained by proteolyticdigestion of 2 mg of MKN45 cell lysate was stored at −80° C. The samplewas dissolved in 200 μL of the binding buffer containing 1M KCl and 100mM Tris-HCl pH 8.0 in deionized water. The sample was dissolved bypipetting up and down several times and subsequently centrifuged at14000 RPM on a tabletop centrifuge for 5 minutes at 4° C. 2 μL of thesupernatant was measured on DS-11 FX+ spectrophotometer/fluorometer(DeNovix) using the Absorption 280 application provided with theinstrument. Two to three replicate measurements were performed on eachsample. The measured sample absorption at 280 nm was subsequentlyconverted into the protein concentration using the formula 1OD A280=1mg/ml and the total protein (peptide) amount in the sample wascalculated by multiplying the protein concentration by the samplevolume.

While preparing two or more samples for MS analysis, each sample wasmeasured as described above and the protein (peptide) content of eachsample was made to be approximately equal by adjusting the samplevolume.

Example 5 Multiplexed Affinity Binding of Peptide Analytes to a BeadArray

1 mg of the digested MKN45 cell lysate dissolved in 200 μL of thebinding buffer was mixed with 1 pmol of the synthetic peptideGSPSGLRPRGY[pT]ISDSAPSR (SEQ ID NO: 5) and the mixture transferred intoa single well of an EPPENDORF® 96 well plate. The well was subsequentlysealed using PARAFILM® tape to prevent solvent evaporation. Magneticmicrobeads conjugated to #4370 and #3617 antibodies were added to thelysate. The 96 well plate was inserted into the EPPENDORF® Thermomixer Cand the microbeads incubated with the lysate for at least 3 hours and atmost 24 hours at 4° C. and shaking at 1200 RPM. Individual reactivesites of the bead array were subsequently measured by MALDI TOF MS.Post-data acquisition calibration of each mass spectrum was performedusing m/z values of the singly- and doubly-charged ions of the syntheticpeptide. Signals from both the Erk1 and Erk2-derived peptides and fromthe synthetic peptide were detected in the mass spectra. It was observedthat incubation times longer than 3 hours provided more stable ratios ofsignals from a specific Erk1 or Erk2-derived peptide to the signal fromthe synthetic peptide, indicating that kinetics of binding of the Erk1and Erk2-derived peptides and the synthetic peptide to individualreactive sites of the bead array may require more than 3 hours to reachthe equilibrium.

Example 6 Reusable Microarray Substrate Containing a Chamfer

A reusable microarray substrate was produced by affixing a siliconerubber gasket, which contained an array of through holes, to a cleanflat surface of an ITO-coated glass microscope slide. The gasket wasmade of a standard grade polydimethylsiloxane (PDMS), the material foundin press-to-seal SILICONE ISOLATORS™ products from Grace Bio-Labs (Bend,Oreg.). The custom gaskets were produced by Grace Bio-Labs. Red siliconegaskets had dimensions of 24 mm×74 mm×0.5 mm and contained a square gridarray of 26×88 microwells. An internal diameter of each microwell was0.5 mm with adjacent microwells separated by a distance of 0.3 mm (0.8mm measured as center-to-center). An area of approximately 1.5 mmbetween peripheral wells and the edges of the gasket contained no wells.Clear silicone gaskets had dimensions of 24 mm×74 mm×0.25 mm andcontained a square grid array of 48×148 microwells throughout the gasketarea. An internal diameter of each microwell was 0.25 mm with adjacentmicrowells separated by a distance of 0.5 mm measured ascenter-to-center.

Silicone gaskets received from the manufacturer were trimmed to create avisual marking in the form of a chamfer (FIG. 3 ) to distinguish the“top” surface of the gasket, which may be repeatedly exposed to theMALDI matrix solution, from the “bottom” surface, which contacts themicroscope slide and should not be exposed to the matrix solution. Thevisual marking, e.g. the asymmetrical shape of the silicone gasket wasused to properly orient the gasket relative to the microscope slide whenassembling a new microarray substrate and also when assembling amicroarray substrate using a gasket that has been previously exposed tothe MALDI matrix solution. In this Example, the bottom surface of thesilicone gasket shown in FIG. 3 is suitable for bonding to a microscopeslide when the gasket is horizontally oriented and the chamfer is foundin the upper left corner of the gasket.

It was experimentally verified that while adhesive properties of the“top” surface of the silicone gasket had noticeably changed following anexposure to the MALDI matrix solution, no silicone contamination ofsamples containing bead-eluted analytes was detected, even afterrepeatedly using the gasket.

Further in reference to FIG. 3 , it is noted that the “bottom” surfaceof the gasket is sufficiently smooth throughout its entire area,possesses self-adhesive properties and forms a fluidic seal when bondedto the solid support (the microscope slide). A portion of the “top”surface contains burrs that surround openings into individual microwells(through holes) and is therefore not self-adhesive within an area thatcontains the openings into microwells. A portion of the “top” surface,which is located between peripheral microwells and edges of the gasket,lacks burrs, is smooth, self-adhesive and capable of forming a fluidicseal. Approximate locations of the corresponding portions of the “top”surface are labeled by arrows in FIG. 3 .

Example 7 Microarray Substrate Containing a Layer of MALDI Matrix

A reusable microarray substrate containing a silicone rubber gasketattached to a gold-coated microscope slide was prepared as described inthe previous Example. The microarray substrate was subsequently coatedwith a layer of CHCA MALDI matrix according to the previously describedmethod of matrix deposition using iMatrixSpray. The matrix solutioncontained 10 mg/ml CHCA in 50% acetonitrile and did not contain a strongacid, such as TFA or FA. The pH of the matrix solution was near 6. 10cycles of matrix deposition produced a sufficiently dense surface layerof CHCA matrix that fully covered the bottom and sidewalls of individualmicrowells as well as a surface area of the gasket between openings intomicrowells. The size of individual CHCA matrix crystals was less than 50microns. Because of the absence of a strong acid in the matrix solution,hydrating the matrix-coated microarray substrate with deionized waterkept the pH of the liquid medium on the surface and inside individualmicrowells near 6.

A microarray substrate containing a surface layer of MALDI matrix hasseveral useful properties. Specifically, the matrix layer prolongsevaporation of water from the microwells, allows easier removal ofdroplets of water from the upper surface of the microarray substrate andmay improve incorporation of bead-eluted peptide analytes into MALDImatrix, similarly to the sandwich method of MALDI sample preparation.

The protocol for eluting analytes from bead arrays using a pre-coatedmicroarray substrate is similar to the protocol using a regularmicroarray substrate, although fewer cycles of matrix application may berequired with the former. Because the pre-coated matrix layer does notcontain a strong acid, the bead-bound analytes are not eluted from abead array until the acidic pH matrix solution is applied.

This Example describes a method of preparing a bead array for analysisby mass spectrometry that includes the following steps: providing amicroarray substrate, which includes an elastomer member (siliconerubber gasket) that is bonded to a flat surface solid support(gold-coated microscope slide), the microarray substrate containing anarray of microwells (through holes within the silicone gasket), thebottom surface and the sidewalls of individual microwells coated with asurface layer of MALDI matrix (CHCA, SA or other), which does notcontain a strong acid; assembling a bead array by placing beads in anaqueous solution (e.g. deionized water) into individual microwells ofthe microarray substrate such that pH of the aqueous solution insideindividual microwells remains near neutral; and once the bead array isassembled, contacting the bead array containing the aqueous solutionwith an aerosol containing acidic MALDI matrix solution, wherein theaerosol contacting step is initiated more than 3 minutes, more than 5minutes or more than 10 minutes after the bead array is assembled.Despite the time delay, the beads located inside the microwells remainhydrated because of the presence of the matrix surface layer in themicroarray substrate, which slows down evaporation of water from themicrowells.

Example 8 Eluting Analytes from a Bead Array

A bead array containing affinity bound peptides was produced aspreviously described and subsequently prepared for analysis by MALDI TOFMS. The beads were placed into individual microwells of the reusablemicroarray substrate. Prior to placing the beads on the microarraysubstrate, all microwells were filled with deionized water.

The fabricated bead array was briefly centrifuged using a customdesigned slide spinner at the speed of 6000 RPM to remove droplets ofbulk water from the gasket surface without displacing the beads from themicrowells. The bead array was then placed into the matrix sprayer andrepeatedly exposed to an aerosol containing a solution of MALDI matrix,which was delivered using a custom designed spraying protocol.

The matrix solution spraying protocol was designed to adjust the rate ofmatrix solution deposition according to the ambient humidity level asmeasured by a hygrometer, such that the rate of the matrix solutiondeposition into the microwells is approximately equivalent to the rateat which the solvent of the matrix solution is escaping from themicrowell via evaporation. For ambient humidity levels between 35% and65% of relative humidity and indoor temperature between 20 and 28° C.,the matrix deposition parameters of iMatrixSpray were set as follows:Height: 60 mm; Line Distance: 0.5 mm; Speed: 60 mm/s; Density: 5 μL/cm²;Number of cycles: 10; Delay: 0 sec; Spray area width: 80 mm; Spray areadepth: 30 mm. For ambient humidity levels between 15% and 35%, the speedparameter was changed to 90 mm/s, the other parameters remaining thesame. For ambient humidity levels between 65% and 85%, the speedparameter was changed to 40 mm/s and the line distance parameter to 0.3mm, the other parameters remaining the same. In all cases, the MALDImatrix solution contained 5 mg/ml of α-hydroxy cinnamic acid (CHCA),0.4% (v/v) of TFA and 50% (v/v) of acetonitrile.

In order to prevent splattering of the liquid medium from themicrowells, the pressure setting for the carrier nitrogen gas ofiMatrixSpray was set to 0.09 MPa, slightly below the lowest recommendedsetting of 0.1 MPa.

Example 9 Distinct Shape of Microarray Spots Containing Bead-ElutedAnalytes

FIG. 4 is a photograph of a gold-coated microscope slide containing amicroarray of spots produced using the previously described methods. Themicroarray has 88 columns, 26 rows and contains a total of 2288 spots.The fiducial is located in the upper left corner adjacent to the spotX01 Y01. Two larger irregular shape spots that cover an area of severalregular spots are visible in the upper left and lower right corners ofthe microarray. These spots were manually added to the microarray andcontain a calibration standard, namely trypsin-digested bovine serumalbumin (BSA). Two regions of the microarray that contain analytes arelocated within columns X24-X31 and X58-X66, respectively and areidentifiable by the characteristic donut or crescent shape of the spotsformed in locations that previously contained a bead. No spots weredetected that previously contained more than one bead. The two regionscontain analytes obtained from distinct biological samples and areseparated by several rows and/or columns of spots that are devoid ofbiological analytes, e.g. peptides. Spots that did not previouslycontain a bead are identifiable by their uniform pattern of matrixcoverage. The analyte-devoid spots that are located within a regioncontaining bead-eluted analytes have a different density of the matrixcoverage compared to the analyte-devoid spots located in rows and/orcolumns that separate distinct regions. Specifically, analyte-devoidspots located in the area defined by coordinates X58-X66 and Y03-Y08have a visibly distinct density of matrix coverage compared to adjacentspots in columns X67-X88 and rows Y1-Y2. The visually distinctappearance of such spots was also evident when the microarray wasexamined using a video camera of a MALDI TOF mass spectrometer (BrukerDaltonics Autoflex Speed).

The characteristic shape of microarray spots that initially contained abead and therefore contained or were suspected of containing bead-elutedanalytes was used to select such spots for MS analysis and to excludespots that did not contain a bead and therefore were not expected tocontain analytes of interest. For example, spots with coordinates X24Y11and X24Y12 were selected for MS analysis, while spots with coordinatesX24Y07 and X24Y08 were excluded from MS analysis. The process of spotselection involved optically analyzing the microarray, either manuallyor automatically using a photograph or a scanned image of themicroarray. The selected analyte-containing spots were submitted forbatch mode data acquisition using the AutoXecute mode of Autoflex Speedor analyzed individually. In either case, the MS data was acquired froman entire area of each selected spot including inner areas thatcontained little or no matrix.

While the microarray shown here was produced using CHCA MALDI matrix itis noted that other types of MALDI matrix including SA will producesimilar visual effects to enable spot differentiation andclassification.

Example 10 Optically Encoded Microarray

The pattern of analyte-containing spots within the microarray shown inFIG. 4 was used to encode the microarray, that is to unambiguouslyassociate information about the microarray with an optical image of themicroarray. The microarray contains 2288 spots with some spots having acharacteristic donut or crescent shape, other spots having a roundshape. All spots form a square grid. As previously noted, the donut orcrescent-shaped spots contain or are suspected of containing analytes,which were captured on individual reactive sites of a bead array, whileround-shaped spots are blank and not suspected of containingbead-captured analytes. Because locations of analyte-containing spotsthroughout the microarray are random and not identical between differentmicroarrays, the coordinates of such spots may be used to encode themicroarray. The number of possible encoding combinations is determinedby the number of analyte-containing spots and is sufficient to encodethousands of microarrays.

For the exemplary microarray shown in FIG. 4 , a small fraction of thetotal number of spots was used to create an encoding combination. Thecombination included X24Y11, X24Y12, X25Y09, X25Y10 as coordinates of 4donut/crescent-shaped spots and X24Y07, X24Y08, X25Y07, X25Y08 ascoordinates of 4 round-shaped spots. A total of 8 spots out of 2288spots were used to create the encoding combination, which included bothblank and analyte-containing spots. The combination was associated withan information about the microarray, namely time and date of themicroarray manufacture, description of biological samples used for themicroarray manufacture, description of the bead arrays used for themicroarray manufacture, ID and location of a person or a roboticworkstation that produced the microarray and whether the microarray hasbeen previously measured by MS.

The described encoding method is useful for maintaining identity ofmicroarrays as they are being transferred between the sample preparationmodule, e.g. MALDI matrix sprayer and the measurement module, e.g. MALDIMS instrument. It is also useful for keeping track of microarrays asthey are shipped to a different location, e.g. by mail. It is alsouseful for storing microarrays and subsequently retrieving them fromstorage for additional measurements. It is noted that the describedencoding system is compatible with the sample-consuming methods ofanalysis because MS matrix is usually not fully depleted during a singleMS analysis thereby preserving the visual appearance of individual spotsand also because the spots that do not contain analytes may not need tobe measured thereby also preserving their visual appearance in multiplemeasurements.

Overall, this Example describes methods of encoding and decodingmicroarrays, in which a spot that contains or is suspected of containingan analyte is visibly distinct from a spot that does not contain theanalyte, the analyte-containing spot and the analyte-devoid spot havingvisibly distinct distribution of matrix for mass spectrometry withinrespective spots. The microarray encoding method includes steps ofcreating a decoding combination that includes coordinates of at leasttwo spots within the microarray and associating the decoding combinationwith information about the microarray. In an embodiment, at least one ofthe at least two spots is an analyte-containing spot and at least one ofthe at least two spots is an analyte-devoid spot. An example of adecoding combination for the microarray in FIG. 4 , which is based oncoordinates of 8 spots, is X24Y11(A), X24Y12(A), X25Y09(A), X25Y10(A),X24Y07(B), X24Y08(B), X25Y07(B), X25Y08(B), where A denotes ananalyte-containing (Analyte) spot and B denotes an analyte-devoid(Blank) spot. The microarray decoding method includes steps of obtaininga first microarray, obtaining a decoding combination, which containscoordinates of at least two spots within a second microarray and isassociated with information about the second microarray, matchingcoordinates of at least two spots within the first microarray with thecoordinates present in the decoding combination and verifying that thefirst microarray is identical to the second microarray, therebyobtaining information about the first microarray. In an embodiment, atleast one of the at least two spots in the decoding combination is ananalyte-containing spot and at least one of the at least two spots is ananalyte-devoid spot. In an embodiment, the microarray decodingcombination further includes information about localization of thematrix for mass spectrometry within an analyte-containing spot. Forexample, in reference to FIG. 4 , it can be seen that theanalyte-containing spots X24Y11 and X24Y12 have visibly distinctlocalization of the matrix within respective spots.

Example 11 Binding an Analyte to Distinct Reactive Sites of a Bead Array

The sample is 1 mg of Lys-C digested human brain tissue dissolved in 200μL of the binding buffer. The bead array is assembled by individuallypreparing and subsequently combining 6 distinct reactive sites, each ofthe reactive sites containing a capture agent (antibody) that recognizesa specific epitope within the human microtubule-associated protein tau.The entry number and the entry name for tau in the Universal ProteinResource (UniProt) database are P10636 and TAU_HUMAN, respectively. Eachof the 6 distinct reactive sites is provided in triplicate, thereforethe bead array contains a total of 18 reactive sites. The unconjugatedantibodies were purchased from Abcam (Cambridge Mass.) and BioLegend(San Diego Calif.). All antibodies were provided in BSA and azide-freeform. The catalog numbers are Abcam 242345, BioLegend 806503, Abcam244231, Abcam 156623, Abcam 196359 and BioLegend 806404. The procedureof antibody conjugation to the beads was performed as previouslydescribed.

The specificity of each antibody was determined using immunoaffinitycapture of proteolytic fragments of tau on individual beads followed byMS and MS-MS analysis of the captured fragments.

The #242345 antibody captured multiple analytes that correspond tovarious proteolytic fragments of tau between amino acids 50-150including analytes with m/z values (monoisotopic) of 2178.0 (fragment88-109), 2773.3 (fragment 85-112), 2993.3 (fragment 91-120), 3954.8(fragment 88-126), 4400.1 (fragment 103-145) and 4546.1 (fragment93-136). All tau protein sites are numbered according to the canonicalsequence of the isoform PNS-tau containing 758 amino acids, UniProtidentifier P10636-1. It was noted that many of the detected fragmentscontained cleavage sites that were not specific to LysC.

The #806503 antibody captured an analyte with m/z value of 2677.2 thatwas assigned to the acetylated N-terminal fragment of tau containingamino acids 2 through 24.

The #244231 antibody captured an analyte with m/z value of 2391.0 thatwas assigned to the fragment of tau containing amino acids 45 through67.

The #156623 antibody captured analytes with m/z values of 5599.6 and5679.6, which were assigned to the C-terminal fragment of tau containingamino acids 703 through 755, and 2 and 3 phosphorylated sites,respectively.

The #196359 antibody captured multiple analytes with m/z values of3533.7, 3613.6, 3693.6, 3773.6 corresponding to the fragment of taucontaining amino acids 508 through 541 and 1, 2, 3 and 4 phosphorylatedsites, respectively. The #196359 antibody also captured analytes withm/z values of 3661.7, 3741.7 and 33821.7 corresponding to the fragmentof tau containing amino acids 508 through 542 and 1, 2 and 3phosphorylated sites, respectively. The 508-541 fragment contains 0missed cleavage sites for LysC while the 508-542 fragment contains 1missed cleavage site. The #196359 antibody further captured analyteswith m/z values of 2131.9, 2211.9 and 2291.8 corresponding to thefragment of tau containing amino acids 508 through 528 and 1, 2 and 3phosphorylated sites, respectively. The 508-528 fragment containsLysC-specific cleavage site on the N-terminus (Lys) and nonLysC-specific cleavage site on the C-terminus (Arg). According to themanufacturer, the #196359 antibody specifically recognizesphosphorylated Ser519 in human tau.

The #806404 antibody captured analytes with m/z values of 3453.7, 3533.7and 3613.6, which were assigned to the fragment of tau containing aminoacids 508 through 541 and 0, 1 and 2 phosphorylated sites, respectively.The #806404 antibody also captured analytes with m/z values of 3581.8and 3661.8, which were assigned to the fragment of tau containing aminoacids 508 through 542, and 0 and 1 phosphorylated sites, respectively.The 508-541 fragment contains 0 missed cleavage sites for LysC while the508-542 fragment contains 1 missed cleavage site. The #806404 antibodyalso captured analytes with m/z values of 1808.8 and 1888.8, which wereassigned to the fragment of tau containing amino acids 508 through 526,and 0 and 1 phosphorylated sites, respectively. The #806404 antibodyalso captured analytes with m/z values of 2051.9 and 2131.9, which wereassigned to the fragment of tau containing amino acids 508 through 528,and 0 and 1 phosphorylated sites, respectively. The 508-526 and 508-528fragments both contain the LysC-specific cleavage site on the N-terminus(Lys) and non LysC-specific cleavage site on the C-terminus (Arg).According to the manufacturer, the #806404 antibody specificallyrecognizes a fragment of human tau containing amino acids 527 through547.

Contacting the human brain sample with the 6-plex bead array causes thesingly phosphorylated 508-541 fragment of tau (m/z 3533.7) to bind tothe reactive sites containing the antibody #196359 and the antibody#806404 and the 508-541 fragment of tau containing zero phosphorylationsites (m/z 3453.7) to bind only to the reactive site containing theantibody #806404.

Example 12 Incubating Bead Arrays with Different Amounts of a Sample

The first sample is 1 mg of trypsin digested MKN45 cell lysate dissolvedin 200 μL of the binding buffer. The second sample is 50 μg of thetrypsin digested MKN45 cell lysate dissolved in 200 μL of the bindingbuffer. Each of the first and second bead arrays contained 3 replicatesof a reactive site that contained a capture agent for humanvoltage-dependent anion-selective channel protein 1 (VDAC1). The entrynumber and the entry name for VDAC1 in the UniProt database are P21796and VDAC1 HUMAN, respectively. The BSA- and azide-free VDAC1 antibodywas purchased from Abcam, catalog number 240128. According to themanufacturer, the immunogen for the #240128 antibody is a “syntheticpeptide within Human VDAC1/Porin aa 1-100 (N terminal)”.

The first sample was contacted with the first bead array and the secondsample was separately contacted with the second bead array. Individualreactive sites of the first and the second bead arrays were analyzed byMS. In both the first and the second samples, the #240128 antibodycaptured analytes with monoisotopic m/z values of 1173.6 and 2473.1. The1173.6 signal was assigned to the N-terminally acetylated fragment ofVDAC1 containing amino acids 2 through 12. The 2473.1 signal wasassigned to the N-terminally acetylated fragment of related proteinVDAC2 (P45880, VDAC2 HUMAN) containing amino acids 2 through 23. VDAC1and VDAC2 share significant sequence similarity in the N-terminalregion. It was observed that the measured ratio of VDAC1 fragment toVDAC2 fragment was higher in the first sample that contained 1 mg oftotal input peptide compared to the second sample that contained 1/20 ofthe amount of total input peptide of the first sample. This Exampledemonstrates that competitive binding of protein fragments that arederived from different proteins, yet contain an epitope that isrecognized by the corresponding antibody may be probed in the bead arrayformat by contacting identical reactive sites with different amounts ofa same sample.

Example 13 Consecutively Incubating a Sample with Multiple Bead Arrays

The sample is 1 mg of trypsin digested HCT116 human colon cancer celllysate dissolved in 200 μL of the binding buffer. Each of the first andsecond bead arrays contained 3 replicates of a reactive site thatcontained a capture agent for eukaryotic translation initiation factor4E-binding protein 1 (Q13541, 4EBP1_HUMAN). The BSA- and azide-free4E-BP1 antibody was purchased from Cell Signaling Technology, catalognumber 9644. According to manufacturer, “the 4E-BP1 (53H11) Rabbit mAbis produced by immunizing rabbits with a synthetic peptide correspondingto residues surrounding Ser112 of human 4E-BP1”.

The sample was contacted with the first bead array as describedpreviously. After the incubation, an unreacted portion of the sample wassaved and within 24 hrs contacted with the second bead array. In aseparate experiment, the sample was subjected to at least onefreeze-thaw cycle after the incubation with the first bead array andbefore incubation with the second bead array.

Individual reactive sites of the first and the second bead arrays wereanalyzed by MS. In both the first and the second bead arrays, the #9644antibody captured analytes with monoisotopic m/z values of 1468.6 and1496.6. The 1468.6 and 1496.6 signals had nearly identical intensity.The 1468.6 signal was assigned to the C-terminal fragment of 4E-BP1containing amino acids 106 through 118. The 1496.6 signal was assignedto the same C-terminal fragment of 4E-BP1 containing an A107V amino acidsubstitution that causes a +28 Da mass shift. According to the publicdatabase of somatic mutations in cancer, namely COSMIC Cell LinesProject from canSAR of Institute of Cancer Research UK, the HCT116 cellline contains a missense mutation A107V in 4E-BP1. The A107V mutation isessentially unique to HCT116 as it is not found in other commonly usedcancer cell lines

This Example illustrates the concept of saving an unreacted portion of asample and using it to perform multiple sequential immunoaffinityenrichment reactions. The same target (C-terminal fragment of 4E-BP1)was enriched in two consecutive immunoaffinity reactions. The uniquecell line proteomic signature, namely the two equal intensity signalsseparated by 28 Da was used to confirm the identity of the sample asbeing obtained from the HCT116 cell line. It is possible to provide abead array containing additional capture agents that are specific forunique proteomic signatures of other cell lines. Furthermore, thequantitative nature of MS also allows monitoring of contamination of aparticular cell line with a different cell line. In this Example,detecting even a trace of contamination with the HCT116 cell line ispossible by monitoring the appearance of a signal near 1496.6 in aspectrum that contains the wild-type 4E-BP1 signal near 1468.6. As thedynamic range of MALDI MS instruments routinely span 3 orders ofmagnitude, it is possible to detect contamination of a cell line withless than 10% and even less than 1% of another cell line.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thepresent disclosure has been described in connection with the specificembodiments thereof, it will be understood that it is capable of furthermodification. Furthermore, this application is intended to cover anyvariations, uses, or adaptations of the disclosure, including suchdepartures from the present disclosure as come within known or customarypractice in the art to which the disclosure pertains, and as fall withinthe scope of the appended claims.

REFERENCES

www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/protein-biology-application-notes/calculate-number-immobilized-proteins-per-bead-agarose-affinity-supports.html

ThermoFisher Scientific Application Note: A consideration of relativesizes and dimensions of agarose resin beads, antibodies, proteins,chemical modification groups, and affinity ligands by Douglas A.Hayworth, Ph.D.; Greg T. Hermanson, B. S. dated Mar. 3, 2014, websiteaccessed Jul. 4, 2019

What is claimed is:
 1. A method for eluting an analyte from a beadarray, the bead array comprising a bead positioned in a microwell, thebead being associated with an analyte, the microwell containing anaqueous medium, the method comprising the step of repeatedly depositingan elution medium into the microwell, the elution medium comprising adissolved matrix for mass spectrometry (MS) and a solvent, wherein arate, at which the elution medium is being deposited into the microwell,is approximately equivalent to a rate, at which the solvent is escapingfrom the microwell via evaporation, such that the bead is continuouslyexposed to the solvent and a solution that forms in the microwell has ahigher concentration of the dissolved matrix relative to the elutionmedium.
 2. The method of claim 1 wherein the bead is continuouslyexposed to the elution medium for at least 5 minutes.
 3. The method ofclaim 1 wherein a concentration of the matrix in the elution medium isless than 10 mg/ml.
 4. The method of claim 1 wherein the solventcontains more than 0.2% of a strong acid.
 5. The method of claim 1wherein the elution medium is being deposited into the microwell usingconditions that substantially prevent splattering and spilling of theelution medium from the microwell.
 6. The method of claim 1 wherein theelution medium is being deposited using a programmable device capable ofproducing an aerosol and the method further comprises the step ofsupplying at least one value to the programmable device, the at leastone value being selected according to an ambient air humidity level. 7.The method of claim 6 wherein the at least one value is further selectedaccording to at least one of a chemical composition of the analyte, achemical composition of the elution medium, and a chemical compositionof the aqueous medium.
 8. The method of claim 6 wherein the programmabledevice is operably connected to a hygrometer.
 9. A method for eluting ananalyte from a bead array, the method comprising the steps of receivinga bead array, the bead array comprising a bead that is positioned in amicrowell, the bead being associated with an analyte, the microwellcontaining an aqueous medium, depositing a first amount of an elutionmedium into the microwell, the elution medium comprising a dissolvedmatrix for MS and a solvent, and allowing the aqueous medium and theelution medium to form a mixture, the mixture being sufficiently acidicto cause the analyte to dissociate from the bead, depositing a secondamount of the elution medium into the microwell such that aconcentration of the matrix in the mixture exceeds a concentration ofthe matrix in the elution medium and allowing the solvent to evaporatefrom the microwell thereby causing the matrix to co-crystallize with theanalyte.
 10. A method for eluting an analyte from a bead array, themethod comprising the steps of receiving a bead array, the bead arraycomprising a bead that is positioned in a microwell, the bead beingassociated with an analyte, the microwell containing an aqueous medium,depositing an elution medium into the microwell, the elution mediumcomprising a dissolved matrix for MS and a solvent, wherein the aqueousmedium and the elution medium are allowed to form a mixture underconditions that substantially prevent the matrix from precipitating fromthe mixture and providing a sufficient amount of time to allow thesolvent to escape the microwell via evaporation thereby causing thedissolved matrix to precipitate.
 11. The method of claim 10 wherein theamount of time is greater than 10 minutes.